In recent years a number of immunoassay techniques have been developed for the measurement of clinically important ligands. Typically a competitive binding immunoassay utilizes a conjugate of a labeling substance and a binding component which participates in a binding reaction to produce two species of labeled complexes a bound species and a free species. The relative amounts of the labeled complexes are a function of the amount of the ligand to be detected in the test sample.
Where the labeling substance in the bound species and in the free species are substantially indistinguishable by the means used to measure the labeling substance the bound and the free species must be physically separated. This type of assay is referred to as heterogeneous.
The two most widely used heterogeneous immunoassays are the radioimmunoassay (RIA) and the enzyme linked immunosorbant assay (ELISA). In the RIA, a sample containing an unknown amount of antigen is mixed with a known amount of radiolabeled antigen and antibody. The assay components are allowed to react to near-equilibrium and then the antibody-bound antigen is separated from the unbound antigen. Since sample antigen competes with the labeled antigen for a limited number of antibody binding sites, the more antigen in the sample, the less labeled antigen is in the bound fraction (or the more is in the unbound fraction). This type of assay is generally time-consuming (1-3 hours) and labor intensive.
RIA suffers from two major disadvantages: First, the labeling substance employed is a radioisotope which poses numerous problems associated with handling, storage, and disposal. Second, RIA is performed in a competitive mode (i.e., the analyte and the labeled analyte compete for a limited number of binding sites on the antibody), and, therefore, the antibody affinity constant limits the sensitivity of the assay, typically in the range of 10.sup.-8 M.sup.-1 to 10.sup.-11 M.sup.-1.
ELISA is similar in principle to RIA except that the labeling substance is an enzyme rather than a radioisotope. Other labeling substances have been described in addition to isotopes and enzymes. These include fluorophores coenzymes bioluminescent materials, and enzyme inhibitors. All suffer from the limitation that sensitivity is a strict function of the antibody affinity constant.
Various methods of effecting the separation step in heterogeneous immunoassays are known. These include filtration, centrifugation and chromatography.
U.S. Pat. No. 3,654,090, issued Apr. 4, 1972, to Schuurs et al., describes a noncompetitive heterogeneous immunoassay for human chorionic gonadotropin (HCG) which uses an excess of enzyme-labeled divalent antibody and an immobilized HCG column to accomplish the separation step.
U.S Pat. No. 4,200,436, issued Apr. 29, 1980 to Mochida et al., discloses an immunoassay employing a labeled monovalent antibody in which immobilized antigen (the same antigen as that to be measured) is used to separate the free labeled antibody from the labeled antibody-antigen complex. Since it is primarily the bound fraction which is measured, this assay is usually performed in a competitive mode. Hence, sensitivity is limited by the affinity constant of the antibody when the assay is performed according to the preferred mode.
U.S. Pat. No. 4,098,876 issued July 4 1978, to Piasio et al., discloses a reverse sandwich immunoassay in which the analyte is incubated with labeled antibody prior to incubating with the immobilized second antibody. After separation of the bound, labeled complex from the incubation medium the bound label is measured.
U.S. Pat. No. 4,376,110, issued Mar. 8, 1983, to David et al.. discloses the use of monoclonal antibodies in a two-site sandwich immunoassay format. The preferred mode disclosed involves the measurement of the bound label after separation from the free label.
The preferred mode of operation of the heterogeneous immunoassay techniques described above is to utilize excess primary labeled binding partners and/or excess bound, secondary binding partners to enhance the speed, sensitivity and precision of the assay. When operating in this preferred mode, it becomes necessary to measure the activity of the bound label since there is generally too much free label to allow accurate detections of the small decrease in the amount of free label. The detection of bound label is particularly difficult in some automated analyzers where it is often necessary to introduce the sample to the analyzer in a liquid form.
Materials such as proteins protein-hapten conjugates and specifically antibodies can be adsorbed onto the surface of solid or liquid supports such as polyethylene, polycarbonates, perfluorocarbon polymers, latex particles, glass, and magnetic particles with polystyrene being preferred. In general this adsorption is considered to be irreversible even in the presence of surfactants, chaotropes denaturants such as 8M urea or guanidine hydrochloride. [Methods in Enzymology XL, 149 Ed. K. Mosbach, Academic Press (1976): Morrissey B. W., Annals of the New York Academy of Sciences, 283, 50-64 (1977)].
While proteins are thought to be irreversibly bound under many aqueous conditions, some solvents may cause desorption from hydrophobic surfaces. For example, dimethylsulfoxide (DMSO) and tetrahydrofuran (THF) have been used in hydrophobic chromatography to desorb proteins that were adsorbed from aqueous solution onto hydrophobic supports, presumably by changing the van der Waals or London forces between the protein and the support. These solvents, however, often attack and dissolve organic supports and, at least in the case of DMSO, denature many proteins such as enzymes. [J. Colloid and interface Science, Vol. 76, No 1. 254-255 (1980); C. J. Van Oss et al., Sep. Purif. Methods, Volume 7, 245 (1978); C. J. van Oss et al., Sep. Sci. Technol., Volume 14 305 (1979)].
When the walls of a solid support, such as provided by tubular containers formed of polyethylene, polycarbonates, perfluorocarbonpolymers or glass or the like, are coated with protein-hapten conjugates or antibodies such materials are bound to the surface of the solid support. As described by Luddy, these materials can be released from the solid support surface after they have formed a labeled complex during the immunoassay. The release is accomplished by the use of a release agent which may be monovalent or divalent salts or an organic base. Even using this release procedure, immunoassays of this type tend to be inaccurate due to (a) lack of proper timing of the reaction, (b) improper incubation temperatures and (c) the relatively low diffusion rates of the biological material of interest in the analyte. This biological material must become bound to the proteinaceous binding partner of the analyte which can only occur by contact. Timing is particularly important because of the capture reaction is not allowed to go to completion. Because of the volume of the analyte, proper contact can be difficult to achieve thus rendering accurate timing of the reaction a problem if uniform results are to be obtained. Also the reactions must be terminated at a controllable time. This is often accomplished by introducing a quenching reagent to completely stop the reaction, but this can interfere with the assay and add to the problem of separating the bound and unbound components.
U.S. Pat. No. 3,801,467 describes an apparatus for providing temperature gradients for the culturing of microorganisms. A central thermal conductive bar is positioned to contain a plurality of test tubes glass rods, petri dishes and any other growth chamber necessary. One end of the bar is connected to a heat source and the other a cooling source to provide a thermal gradient along the length of the bar. This bar is rotatable about its axis via a crank/motor. There is no mention of quenching the reaction.
U.S. Pat. No. 3,535,208 describes an apparatus for providing a temperature gradient for culturing microorganisms. In this device however a shaker table is used external to the gradient box. Fluid is circulated through the two triangular shaped volumes - the fluids are at different temperatures creating a linear gradient along the length of the box. There is no mention of quenching the reaction.
U.S. Pat. No. 3,832,532 describes a method and apparatus for testing antibody susceptibility. Included in the device, is the incubator/shaker apparatus shown in FIGS. 22 and 23. Three racks 54 hold ten cuvettes in the chamber that is held at 36.degree. C. These trays are rotated on the platform 58 at 220 rpm at a 3/4 inch amplitude. There is no capability for automatic quenching.
There is a need for an improvement of heterogeneous immunoassays wherein the time temperature and contact rate of the reaction components are better controlled.